丙泊酚对脂多糖诱导的MHCC97H细胞增殖、凋亡和迁移的影响

Effect of propofol on proliferation,apoptosis and migration of MHCC97H cells induced by lipopolysaccharide

目的探究丙泊酚对脂多糖(LPS)诱导的人肝癌细胞增殖、凋亡、迁移、炎症因子的影响及其机制。方法体外培养人肝癌MHCC97H细胞系,分为对照组、LPS组、丙泊酚低剂量组、中剂量组、高剂量组和中剂量+BAY 11-7082组。对照组细胞不做干预,LPS组以1μg/ml LPS处理,低剂量组、中剂量组、高剂量组分别以1μg/ml LPS和12.5,25,50μmol/L丙泊酚同时给药处理,中剂量+BAY 11-7082组以1μg/ml LPS、25μmol/L丙泊酚和10μmol/L NF-κB通路抑制剂BAY 11-7082同时给药共培养处理,均干预24 h。采用酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)表达,活细胞计数(CCK-8)检测细胞活力,5-乙炔基-2′脱氧尿嘧啶核苷(EdU)检测细胞增殖,Hoechst 33258染色法检测细胞凋亡,Transwell小室检测细胞迁移能力,蛋白免疫印迹(WB)法检测细胞周期素D1(Cyclin D1)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、IKKα、IκBα、p-IκBα、NF-κB p65、p-NF-κB p65蛋白水平。结果与对照组相比,LPS组细胞炎症因子TNF-α表达水平和细胞活力显著升高(P<0.05);与LPS组相比,中剂量组和高剂量组炎症因子TNF-α表达水平和细胞活力显著降低(P<0.05),其中高剂量组细胞活力较低,故选择中剂量25μmol/L丙泊酚进行后续实验。与对照组相比,LPS组细胞增殖率、迁移数及Cyclin D1、IKKα、p-IκBα和p-NF-κB p65蛋白表达显著升高,细胞凋亡率和Caspase-3蛋白表达显著降低(均P<0.05);与LPS组相比,中剂量组细胞增殖率、迁移数及Cyclin D1、IKKα、p-IκBα和p-NF-κB p65蛋白表达显著降低(P<0.05),细胞凋亡率和Caspase-3蛋白表达显著升高(P<0.05);与中剂量组比较,中剂量+BAY 11-7082组细胞增殖率、迁移数及Cyclin D1、IKKα、p-IκBα和p-NF-κB p65蛋白表达显著降低(P<0.05),细胞凋亡率和Caspase-3蛋白表达显著升高(P<0.05)。结论丙泊酚可显著抑制LPS诱导的人肝癌MHCC97H细胞的炎症、增殖及迁移,诱导MHCC97H细胞凋亡,其作用机制可能与抑制NF-κB通路的信号转导相关。

Objective To explore the effects of propofol on lipopolysaccharide(LSP)-induced cell proliferation,apoptosis,migration,and inflammatory factors in human hepatoma cells and its possible mechanism.Methods Human hepatoma MHCC97H cells were cultured in vitro and divided into control group(no treatment),LPS group,low dose group,medium dose group,high dose group and medium dose+BAY 11-7082 group.MHCC97H cells were treated with 1μg/ml LPS for 24 h in LPS group,and MHCC97H cells were co-cultured with 1μg/ml LPS and 12.5,25,50μmol/L propofol for 24 h in low-dose group,medium dose group and high dose group.MHCC97H cells were co-cultured with 1μg/ml LPS,25μmol/L propofol and 10μmol/L NF-κB pathway inhibitor BAY 11-7082 for 24 h in medium dose+BAY 11-7082 group.Tumor necrosis factor-α(TNF-α)expression was detected by enzyme-linked immunosorbent assay(ELISA),cell viability was detected by cell counting kit-8(CCK-8),cell proliferation was detected by 5-acetylidene-2′deoxyuracil nucleoside(EdU),and cell apoptosis was detected by Hoechst 33258 staining.Transwell chamber was used to detect the cell migration ability,and Western blot was used to detect the protein levels of Cyclin D1,Caspase-3,IKα,IκBα,p-IκBα,NF-κB p65,p-NF-κB p65.Results Compared with control group,the expression level of TNF-αand the cell viability in LPS group were significantly increased(P<0.05);compared with LPS group,the expression of inflammatory factor TNF-αand the cell viability in medium dose group and high dose group were significantly decreased(P<0.05),and the cell viability was lower in high dose group,thus 25μmol/L propofol was chosen for subsequent experiments.Compared with control group,the cell proliferation rate,the migration number and the protein expressions of Cyclin D1,IKKα,p-IκBαand p-NF-κB p65 were significantly increased in LPS group(P<0.05),while the apoptosis rate and Caspase-3 protein expression were significantly decreased(P<0.05);compared with LPS group,the cell proliferation rate,the migration number,Cyclin D1,IKKα,p-IκBαand p-NF-κB p65 protein expressions were significantly decreased in medium dose+BAY 11-7082 group(P<0.05),while the cell apoptosis rate and Caspase-3 protein expression were significantly increased(P<0.05).Compared with medium dose group,the cell proliferation rate,the migration number,Cyclin D1,IKKα,p-IκBαand p-NF-κB p65 protein expressions were significantly decreased in medium dose+BAY 11-7082 group(P<0.05),while the cell apoptosis rate and Caspase-3 protein expression were significantly increased(P<0.05).Conclusion Propofol can significantly inhibit LPS-induced inflammation,proliferation and migration of human hepatoma MHCC97H cells,and induce the apoptosis of MHCC97H cells,which may be related to the inhibition of NF-κB pathway signaling transduction.